The decision of whether or not to initiate the cell cycle is crucial to the control of cell proliferation. For the yeast Saccharomyces cerevisiae, cells that have executed the late G1 event(s) termed "Start" are committed to initiate DNA synthesis, to form a bud, and to divide. The execution of Start seems to require some threshold level of G1 cyclin/CDC28 kinase activity, which is determined in large part by the rate and levels at which the G1 cyclin RNAs accumulate during late G1. However, very little is known about the mechanisms that determine if G1 cyclin RNAs accumulate and the rate at which G1 cyclin RNAs accumulate. Many cell cycle events are controlled by the regulation of the phosphorylation of certain cell cycle substrates. The SIT4 gene, which encodes the catalytic subunit of a serine/threonine protein phosphatase, is required during late G1 for the execution of Start, for bud formation, for the initiation of DNA synthesis, and for spindle pole body duplication. SIT4 is required for the execution of Start because it is required for the expression of the CLN1 and CLN2 G1 cyclin genes. If CLN2 is expressed from a SIT4-independent promoter, the cells can initiate DNA synthesis in the absence of SIT4. However, independently of CLN2 expression, SIT4 is also required for bud formation. During most of G1, SIT4 exists mostly as monomeric uncomplexed SIT4. Very close to the time SIT4 is required for the execution of Start and for bud formation, SIT4 associates in separate complexes with two high molecular weight proteins, p155 and p190 (the complexes dissociate at about midmitosis). We propose that p155 and p190, like the high molecular weight targeting subunits of type 1 phosphatases, may target SlT4 to specific substrates during late G1. Therefore, SlT4 is a very good candidate for playing a critical role for the control of G1 cyclin expression (which is required for Start and subsequent DNA synthesis) and for bud formation (or polarization of cell growth). The specific aims of this proposal are: A) to determine the specific functions of the p155/SIT4 and p190/SIT4 complexes and their roles in the individual functions of G1 cyclin expression and bud formation; B) to determine how the association of p155 and p190 with SIT4 is cell cycle regulated. If p155/SlT4 and p190/SlT4 are the active forms of SlT4 for G1 cyclin expression and bud formation, then the signals that direct association of p155 and p19O with SlT4 regulate G1 cyclin expression and bud formation; and C+D) to determine the phospho-proteins that require dephosphorylation by SlT4 (or G1 cyclin accumulation (aim C) and for bud formation (aim D). These experiments should increase our understanding of how cells commit to enter the cell division cycle.